In agricultural biotechnology, plants can be modified according to one's needs. One way to accomplish this is by using modern genetic engineering techniques. For example, by introducing a nucleic acid molecule of interest (NOI) into a plant, the plant can be specifically modified to express a desirable phenotypic trait. For this, plants are transformed most commonly with an expression cassette that comprises a promoter, a nucleic acid molecule of interest, and a terminator.
Plant gene based expression cassettes typically control spatial and temporal transcription, an important aspect of trait gene development. However, they are often not capable of producing adequate trait protein, limiting and sometimes eliminating their utility. Many factors affect expression of nucleic acids including chromatin structure, transcription efficiency, transcription factors, mRNA stability and regulatory factors such as promoters, enhancers and silencers. In addition, introns are known to affect expression levels in eukaryotes and in some cases may have a larger influence than, for instance, promoters. (Rose et al. Plant Cell 20:543-551 (2008)). Thus, for example, efficiently spliced introns boost expression more than 10-fold while others have little or no effect (Id.). The first intron of maize shrunken-1 gene has been shown to increase gene expression 1000 fold in maize protoplasts (Maas et al. Plant Mol. Biol. 16(2):199-207 (1990)) and the first intron of rice rubi3 enhanced gene expression about 3-fold in stable transgenic rice (Lu et al. Mol. Genet. Genom. 279(6):563-572 (2008)). The effect of introns on increasing nucleic acid expression is termed intron-mediated enhancement (IME) (Id.). Despite the significant influence of introns on the expression of nucleic acids, little is known about the mechanism IME. Further, only a few introns (mostly first introns) have been evaluated experimentally and are known to enhance gene expression in plants. Little is known about the effect of terminator sequences.
Important aspects of the present invention are based on the identification of introns and terminators that can enhance expression of a NOI when substituted into an expression cassette as a heterologous sequence.